Application of rice OsKNOX8 gene in regulating salt tolerance

Abstract

The application provides application of a rice OsKNOX8 gene in regulation of salt tolerance. The gene OsKNOX8 is a DNA molecule as described in 1) or 2) below: 1) a DNA molecule with a genomic sequence as shown in SEQ ID NO. 1; 2) a DNA molecule with a CDS sequence as shown in SEQ ID NO. 2; 3) a DNA molecule hybridizing with the DNA sequence defined in 1) or 2) under stringent conditions and encoding the protein. The application provides genetic engineering application of the gene OsKNOX8 in regulation of rice salt tolerance, specifically, knocking out the aforementioned gene OsKNOX8 to improve rice salt tolerance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering, and particularly relates to a salt-tolerant related gene OsKNOX8 of rice, and a coding protein and application thereof. BACKGROUND

[0002] With the growth of the world population and the development of industrialization and urbanization, arable land resources are decreasing. At the same time, soil salinization caused by improper cultivation and use also reduces the productivity of arable land, further exacerbating the problem of food security. The high content of salt and alkali components in saline soil changes the physical and chemical properties of the soil, causing soil compaction, decline in fertility and many other problems, leading to soil degradation. Soil salinization not only threatens the sustainable development of global agricultural ecosystems, but also causes a decrease in biodiversity and destruction of regional ecological balance. China has 100 million hectares of saline-alkali land, ranking third in the world, of which about one-third has development and utilization potential, which is an extremely important reserve arable land resource. Rice is the first choice for food crops for the improvement of coastal beaches and saline-alkali land, but rice has poor salt tolerance, and high salt environments seriously harm the growth and development of rice and yield. Therefore, in-depth analysis of the damage caused by salt stress to rice and the physiological and molecular mechanisms of rice in response to salt stress, screening and breeding of salt-tolerant rice varieties, have important significance for the development and utilization of coastal beaches and saline-alkali land and the promotion of sustainable agricultural development.

[0003] Under salt stress, the growth and development of rice will be significantly inhibited, and tissue growth and organ differentiation will be slowed down, and the inhibitory effect will be more obvious with the increase of salt concentration. Rice seeds subjected to salt stress during the seed germination stage not only have limited physiological water absorption, but also the membrane structure of cells during the water absorption process is damaged, leading to blocked seed germination, reduced germination rate and uneven germination. The seedling stage is the most sensitive period of rice to salt stress, and rice seedlings under salt stress show symptoms such as growth inhibition, old leaf death and decreased green leaf area. Salt stress during the tillering stage of rice will inhibit tillering and leaf growth, reduce the tillering capacity of single plant, thicken the stem, and cause leaf yellowing and even death, and reduce plant height. The booting stage is another period of rice sensitivity to salt stress in addition to the seedling stage, and salt stress will cause the booting stage of rice to be delayed, the glume flower to be severely degraded, and the seed setting rate to be reduced, which will seriously affect the yield. In addition, salt stress will also cause changes in seed grain fatty acids, minerals and nutrients, affecting rice quality.

[0004] The physiological regulation mechanism of rice salt tolerance includes osmoregulation, antioxidant system regulation, hormone regulation, etc. Under salt stress, the osmotic potential of the external environment decreases, and the rice root system has difficulty in absorbing water, resulting in osmotic stress. In order to reduce its own water loss and be able to absorb water from the outside, rice responds to osmotic stress through two osmoregulation mechanisms: one is to absorb K + , Ca 2+The first is to produce inorganic ions such as proline, betaine, and soluble sugars; the second is to synthesize organic osmotic regulating substances such as proline, betaine, and soluble sugars. Under salt stress, rice produces and accumulates a large amount of reactive oxygen species, which leads to membrane lipid peroxidation, increased malondialdehyde content, cell damage, and oxidative stress. Rice has an antioxidant system that scavenges reactive oxygen species, including enzymatic defense systems and non-enzymatic defense systems. The enzymatic reaction system mainly includes superoxide dismutase, catalase, ascorbate peroxidase, etc., while the non-enzymatic defense system mainly includes glutathione, ascorbic acid, carotenoids, etc. When rice is exposed to a high-salt environment, the plant produces a variety of plant hormones to jointly regulate the salt stress response to cope with the damage of salt stress to rice growth and development. Hormones that help improve rice salt tolerance mainly include abscisic acid, ethylene, brassinolide, etc., which participate in the rice response to salt stress through complex signal transduction pathways.

[0005] Salt tolerance in rice is a quantitative trait controlled by multiple genes and has a complex genetic basis. Breeding salt-tolerant rice varieties using traditional breeding methods is challenging and progress has been slow. Identifying key genes for salt tolerance in rice and elucidating their mechanisms of action will facilitate rapid genetic improvement of salt tolerance using molecular breeding techniques. However, few of the cloned rice salt-tolerance genes have been effectively applied in rice breeding. Further research is needed to identify key salt-tolerance genes and lay the foundation for breeding new salt-tolerant rice varieties. Summary of the Invention

[0006] In order to overcome the above technical problems existing in the prior art, the present invention provides the genetic engineering application of rice OsKNOX8 gene in regulating rice salt tolerance.

[0007] The technical solution of this patent is as follows:

[0008] Application of the rice salt tolerance-related gene OsKNOX8, or the protein encoded by the gene OsKNOX8, or a knockout vector for the gene OsKNOX8, or primers for amplifying the gene OsKNOX8 in genetic engineering for regulating rice salt tolerance;

[0009] The gene OsKNOX8 is the DNA molecule described in 1) or 2) below:

[0010] 1) A DNA molecule having a genomic sequence as shown in SEQ ID NO. 1;

[0011] 2) A DNA molecule whose CDS sequence is shown in SEQ ID NO.2.

[0012] Furthermore, knocking out the aforementioned gene OsKNOX8, or reducing the expression level of the protein encoded by the gene OsKNOX8, or transferring the knockout vector of the gene OsKNOX8 into rice can improve the salt tolerance of rice.

[0013] Furthermore, the amino acid sequence of the protein encoded by the gene OsKNOX8 is shown in SEQ ID NO.3.

[0014] Furthermore, the knockout vector of the gene OsKNOX8 utilizes CRISPR-Cas9 technology to edit the gene in rice, thereby knocking out the aforementioned gene OsKNOX8 in the rice plant, rendering the gene nonfunctional.

[0015] Furthermore, the knockout vector of the gene OsKNOX8 is a CRISPR-Cas9 vector targeting the target site shown in SEQ ID NO.6.

[0016] Furthermore, the primers for amplifying the gene OsKNOX8 are selected from Primer1 shown in SEQ ID NO.4 and Primer2 shown in SEQ ID NO.5, and the Primer1 / Primer2 are used to amplify the CDS fragment of the OsKNOX8 gene, or Primer5 shown in SEQ ID NO.9 and Primer6 shown in SEQ ID NO.10, and the Primer5 / Primer6 are used to amplify the mutation target region fragment.

[0017] Beneficial effects:

[0018] This study, published in Nature Communications, for the first time, discovered the link between the rice gene OsKNOX8 and rice salt tolerance. Inhibiting the expression of the gene encoding the protein can improve plant salt tolerance, thereby enabling the cultivation of salt-tolerant transgenic plants. The protein and its encoding gene can be used for plant genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The mutation site of the OsKNOX8 gene and its flanking sequences in the OsKNOX8 gene mutants (osknox8-1 and osknox8-2) are shown.

[0020] Figure 2 The seedling phenotypes of wild-type Nipponbare (WT) and OsKNOX8 gene mutants (osknox8-1 and osknox8-2) under salt stress.

[0021] Figure 3 Seedling survival rates of wild-type Nipponbare (WT) and OsKNOX8 gene mutants (osknox8-1 and osknox8-2) under salt stress. DETAILED DESCRIPTION

[0022] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The reagents and kits used in the following examples, unless otherwise specified, were purchased from conventional biological reagent companies.

[0023] Example 1: Cloning of the coding region sequence (CDS) of the rice OsKNOX8 gene

[0024] The CDS fragment of the OsKNOX8 gene was amplified by PCR using the cDNA of rice cultivar Nipponbare seedling leaves as template using the primer pair consisting of Primer1 and Primer2.

[0025] Primer1:5'-ATGGAGAGCTTCGCCAGTCTCGCA-3' (SEQ ID NO.4);

[0026] Primer2: 5'-TCAAGACCCGAGCCGGTACATGCCG-3' (SEQ ID NO. 5).

[0027] The PCR reaction system (50 μl) consisted of 1 μL template cDNA (200 ng / μL), 1.5 μL Primer 1 (10 μM), 1.5 μL Primer 2 (10 μM), 5 μL 10× PCR Buffer for KOD-Plus-Neo, 5 μL 2 mM dNTPs, 3 μL 25 mM MgSO₄, 1 μL KOD-Plus-Neo (1 U / μL), and 32 μL ddH₂O. PCR amplification was performed in a Bio-Rad T100 PCR amplifier. The PCR protocol was as follows: 94°C denaturation for 2 min; 35 cycles of 98°C denaturation for 10 s, 60°C annealing for 30 s, and 68°C extension for 2 min; 5 min extension at 68°C; and storage at 25°C.

[0028] The PCR product was recovered and purified using a DNA purification kit (Nanjing Novozymes Biotechnology Co., Ltd.) and ligated to the pEASY-Blunt expression vector (Beijing Quanshijin Biotechnology Co., Ltd.). The ligation product was transformed into Escherichia coli DH5α competent cells (Baoriyi Biotechnology (Beijing) Co., Ltd.), and positive clones were selected for sequencing (Beijing Qingke Biotechnology Co., Ltd.).

[0029] The sequencing results showed that the CDS fragment of the OsKNOX8 gene obtained by PCR amplification had the nucleotide sequence shown in SEQ ID NO.2 and encoded a protein consisting of 341 amino acid residues shown in SEQ ID NO.3.

[0030] Example 2: Construction of transgenic rice plants expressing the OsKNOX8 gene mutant

[0031] 1. Construction of OsKNOX8 gene knockout vector

[0032] Based on the OsKNOX8 genomic sequence (SEQ ID NO. 1), CRISPR-Cas9 sgRNA targets were designed using the CRISPR-P V2.0 website (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target sequence selected for constructing the OsKNOX8 gene editing vector was GCTGCCGCGCCGACGACTCC (SEQ ID NO. 6).

[0033] Primer3 and Primer4 were synthesized based on the target sequence, and this primer pair was annealed to generate a double-stranded DNA molecule with sticky ends. This double-stranded DNA molecule was ligated with Bsa I-cleaved linearized pOs-sgRNA vector (the vector construction method is described in the paper "Targeted mutagenesis in rice using CRISPR-Cas system") using T4 DNA ligase. The resulting ligation product was transformed into DH5α Escherichia coli. Positive clones were identified by colony PCR, and the plasmids were extracted and sequenced. Sequencing results showed that a recombinant vector containing the sequence shown in SEQ ID NO. 6 was obtained and named pOs-sgRNA-OsKNOX8t.

[0034] Primer3:5'-ggcaGCTGCCGCGCCGACGACTCC-3' (SEQ ID NO.7);

[0035] Primer4:5'-aaacGGAGTCGTCGGCGCGGCAGC-3'(SEQ ID NO.8)

[0036] The pOs-sgRNA-OsKNOX8t plasmid was mixed with the pH-Ubi-cas9-7 vector (the vector construction method is described in the literature "Targeted mutagenesis in rice using CRISPR-Cas system"); the LR reaction was performed using the Gateway kit (Invirogen), and Escherichia coli DH5α was transformed; positive clones were identified by colony PCR, and the plasmid was extracted and sequenced to obtain the plasmid pH-Ubi-cas9-7-OsKNOX8t containing the sequence shown in SEQ ID NO.6.

[0037] II. Obtaining of the Recombinant Agrobacterium

[0038] Mix pH-Ubi-cas9-7-OsKNOX8t with Agrobacterium EHA105 competent cells, incubate on ice for 5 min, transform pH-Ubi-cas9-7-OsKNOX8t into Agrobacterium EHA105 competent cells by electroporation (1500V, 5ms), obtain the recombinant strain, and name the correct recombinant strain identified by colony PCR as EH-pH-Ubi-cas9-7-OsKNOX8t.

[0039] III. Obtaining of Transgenic Plants

[0040] Transform the above-mentioned recombinant Agrobacterium strain into rice variety Nipponbare, and the specific method is as follows:

[0041] (1) Culture the EH-pH-Ubi-cas9-7-OsKNOX8t strain at 28°C, 200 rpm overnight until OD 600 saturation; inoculate the bacterial solution into new YEP liquid medium at a ratio of 1:100, and suspend culture at 28°C, 200 rpm until OD 600 = 0.6-0.8, collect the bacterial cells; mix 30 mL of AAM liquid medium (containing 30 μL of 1000x AS (acetyl-syringone)) with the bacterial cells gently to make OD 600 = 0.05-0.1.

[0042] (2) Mix the mature embryo somatic callus of Nipponbare cultured for one month with the bacterial solution in step (1), and infect for 90s, absorb the bacterial solution with filter paper, and transfer to 2N6-AS solid medium, and culture at 28°C in the dark for 48-60h.

[0043] (3) Take out the callus in step (2), and wash with sterile water for 8-10 times with constant shaking until the water is clear; wash with sterile water containing 500 mg / L Car (carbenicillin disodium) for 2 times, 10-20 min each time; remove the sterile water, and pour the callus in the centrifuge tube on sterile filter paper to drain for 1-2h.

[0044] (4) Inoculate the callus in step (3) on N6D-S solid medium containing 50 mg / L Hyg (hygromycin B) and 250 mg / L Car, and culture at 28°C for 3-4 weeks for screening.

[0045] (5) Transfer the resistant callus screened to MS-NK solid medium, and culture at 28°C for 3-4 weeks to make the callus differentiate to produce adventitious buds.

[0046] (6) When the adventitious buds grow to 3-4 cm, transfer them to MS-HF rooting medium and grow them at 28°C for 1 week.

[0047] (7) The differentiated rice seedlings are hardened and transferred to the field for growth, and the T0 generation transgenic plants are obtained.

[0048] Example 3: Molecular Identification of Rice OsKNOX8 Gene Mutant Transgenic Plants

[0049] The aboveground part of the seedlings of the T0 generation transgenic plants of the OsKNOX8 gene constructed in Example 2 was sampled, and genomic DNA was extracted as a template. The primer pair consisting of Primer5 and Primer6 was used to PCR amplify the editing target site shown in SEQ ID NO. 6 and the DNA fragments on both sides thereof.

[0050] Primer5:5'-TTTGCTAGGGTTTGTTTGCC-3' (SEQ ID NO.9);

[0051] Primer6: 5'-ATACTCACCAGCGAGCGAAT-3' (SEQ ID NO. 10).

[0052] The obtained PCR products were detected by 1% agarose gel electrophoresis and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. According to the sequencing results, two strains with specific mutations in the OsKNOX8 gene were identified: osknox8-1 and osknox8-2. The DNA sequences of the editing target sites and their flanking sites in these two strains are shown in Figure 2. Figure 1 shown.

[0053] In the osknox8-1 strain, a nucleotide C was inserted in the first exon of the OsKNOX8 gene;

[0054] In the osknox8-2 strain, a nucleotide G was inserted in the first exon of the OsKNOX8 gene;

[0055] In these two mutant strains, mutations in the CDS sequence of the OsKNOX8 gene caused frameshift mutations and premature termination of the encoded protein.

[0056] Example 4: Identification of salt tolerance of OsKNOX8 gene mutant strains at the seedling stage

[0057] The two OsKNOX8 gene mutants identified in Example 3 were self-pollinated for two generations to obtain homozygous mutant lines osknox8-1 and osknox8-2. These two homozygous mutant lines were used together with the Nipponbare wild type to perform a salt tolerance test at the seedling stage.

[0058] (1) Select plump rice seeds, soak them in tap water in a 33°C incubator for 2 days, and germinate them for 1 day. Select seeds with uniform germination, sow them on a 96-well PCR plate without a tube bottom, place them in a black organic glass culture box filled with pure water, and culture seedlings in an artificial climate chamber. The culture conditions are: 14 h light (28°C) / 10 h dark (24°C), light intensity 1000 μmol·m -2 ·s -1 After culturing in pure water for one week, the cells were switched to Kimura B nutrient solution.

[0059] (2) When the rice seedlings grew to the two-leaf and one-heart stage, the nutrient solution was replaced with Kimura B nutrient solution containing 100 mM NaCl for salt stress treatment.

[0060] (3) After 10 days of NaCl treatment, rewater the rice seedlings with Kimura B nutrient solution, which does not contain NaCl. Seven days later, the survival rate of the rice seedlings was calculated. Survival rate = number of surviving seedlings / total number of treated seedlings × 100%.

[0061] The results of salt tolerance identification showed that compared with the wild type (WT) of Nipponbare, the two OsKNOX8 gene mutant strains (osknox8-1 and osknox8-2) showed milder salt damage symptoms such as leaf wilting and drying ( Figure 2 ); After 10 days of salt stress and 7 days of rehydration, the survival rate of osknox8-1 and osknox8-2 seedlings was significantly higher than that of the wild type (WT) ( Figure 3 ).

[0062] The above experimental results show that the OsKNOX8 gene has the function of negatively regulating the salt tolerance of rice seedlings, and knocking out this gene can significantly improve the salt tolerance of rice.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0064] SEQ ID NO.1 OsKNOX8 genome sequence:

[0065] CACTGTTGCTGCTGCTGCTGCTGCTTGAAACACTGAATAGCCTAACCTCTTCATTAATTT

[0066] CTCGCTCTGATTTTGACGTTAGAACAAAGCTAAGATGGGCCATACGTATACTAGGAGGAG

[0067] TACCATCATATTCATCTTCTTTCTAGCTTCCTCTCCTCTTCACATTCCCTGCATCCATCCATC

[0068] TCAACCACCACCACCTCATTTCAGGAGATCATCTCCTCCTTTTCTTTGCTAGGGTTTGTTT

[0069] GCCATAACTACTATCTATCGCCTCTTCTTCACCTCACCTGTCCTGTCGGCCATGGAGAGCT

[0070] TCGCCAGTCTCGCAGGAGGTGGCAGCAGCAGCACCACAGCTCGGTTGCCGGAGCTGATC

[0071] TCGCCGGAGAACCCGGACCACATCTCGCCGCCCCCGTTGCTGTACCAGCTGCTCGCCGG

[0072] CCCGGAGTCGTCGGCGCGGCAGCATGGGCATGATGGGCATCACCACGGCGGCGGTGGCG

[0073] GCGAAGCAGCAGCAGCAGCTGTGCAGGGACAGGTCTCGCCGGCAGGCGCGGAGGCGGC

[0074] GGTCAAGGCCGAGATCATGTCGCATCCACAGTACTCGGCTCTCCTAGCCGCCTACTTAGG

[0075] CTGCAAAAAGGTGTAGTAGCTAAGCTAGCAGATGCATATATTTAAACCCATGCAGATACAG

[0076] ACGACAACACGCCATGTGCTGACCAAGCTCACGGCCGTGCCGGATGTACTGACCAAGTT

[0077] GATTTTGTTGGTTTTTTTATCAGGTCGGGGCGCCGCCGGATGTACTGACCAAGCTCACGG

[0078] CTGTGCCGGCGGCGCAGCAGCTGGACGAAGCCGACGGCCACCCTCGTCGCCGGCACGA

[0079] GCCACAGCGTGATGACGACCCGGATCAGCTCGATCAGTTCATGGTATCAACATATATAGAT

[0080] CATCAACTCCTTTGCTCGCATGCGTGCATATATATAGACAGACACACACACATATATATCCA

[0081] CTGTTCTTCTGATCGATCGACATGGGATCTGATCTTGTCATCACAGGACGCGTACTGCAGC

[0082] ATGCTGACGAGGTACAGGGAGGAGCTGGAGCGGCCGATCCTGGAAGCCGCGGAGTTCTT

[0083] CAGCAGGGTGGAGACGCAGCTCGATTCGCTCGCTGGTGAGTATTTTATACGCCTCTTCTCT

[0084] ACAAAGATTTGCAGCTTGCCATATTGGGAAATGCCGAAATGGTATCTCACTTTATGGAATT

[0085] GGAGTTAGTTTTTTTTTTGTCACCTCTTTGGACTCATGTGGCTGTTGCAGCAGTATTTCTTA

[0086] AGGCTGCTAATCTCAGGCTTTAATATGTTTATTAGGGTGGGGGATAAGGGTTGCATGCATT

[0087] AGATCAGTTTCGTAATAATTGGAGCTTAATTAAGTATACATGACCAGTATGATAAGCATCAT

[0088] GAGGTCACACGGTGAGGCTGCGAGCTGAGCTCCAGGGTTTCCAAATTGCCATATATGCAT

[0089] GGCTCTCCTACTTTCCAGTCTCCACTTCCTCCTGTGCAATACAACGATGGTGTCCTCCCTC

[0090] TGTCGTACAATCTTAATGAATCATACAAAGGAATAATTAATACTGGAGCACATGCAGTTCT

[0091] TTGAACTTGTACCTTGTACTGACGTTATTTTTTTTCCTCTGATTTTCTTTTATTTTTAGAATT

[0092] AAGACCCAACCTTGCTGACATTATTAGCTTCTAATTAAAGAGTTCAGTGCATGCATCACAA

[0093] GTGATGAACTGACAGGAGAAAGCTCTCAGCACGTTCTGTCCCCAATATCTCTGCAGGTCC

[0094] TTGTCTAACCTACATTTTTGCTCTCTTGTCATGTCTTCCGCTGGTAGTCACAGAGAAATGG

[0095] CTAGACGATCTTTGTTTTCAACTTAGACGATTTATGTTAAGATTAAGGGCCAAGAGGTAAA

[0096] TTAAACGAACCATCAAAGTTTGCAGAACATAGTGGCCAAATTGAGGTGTAAGTAACATCT

[0097] AGCTGGTTGAAAACATGAGTGGCGGTGGATGACAGAATGAGTGGCCGGTGGGCCTATTG

[0098] TCATTGTGCCTCCGTGAGTGTAATTGGTTTACCGACCTCAAATCCTGGATCGAGTCTTGGC

[0099] TGTCGGACATGAATGGGCACTGTTAGTACCCTGTGCACTTTGTCTCATGGCCTGGATATAT

[0100] AGTTACTGGAGTGGGGTAAGGATCCAGCTATATATTAGACAATTGATAAGTGTGTATGACC

[0101] ATCTAGTTGTGTCCTTTCAAGTGCAGTTGTTTCCGAACATTATTGAGCACTTTCCGTAGCC

[0102] ATACGAATAGATTTCCAGATCAGAACTCTTTCTTATATATACAGACATCAATCGAGTGCCAA

[0103] ATAATTGAAAGAATGAAAGCAGCATAGTGACCAGCATGCATATCATATTGTGTTTTGTTGA

[0104] AACATAGTTCAATAATTTTGTCTATATGAACTAGTGTAGTTTTTTACACTGGTAATTGCATAT

[0105] ATACAAGGTGGTGTTTCATAAGACGACTAATATGGCAAAATTATGAAATTCACTATCCGTA

[0106] CAAATCCTGGATAATATATAGGAGTACATATATGGAAGAATTATCTGGTTTCTTATATATTTA

[0107] GTCTGCCAAAATGTCATTCAGCTATTCCCCTACCGATGAGGCATTTTTTTTTGTGTCTACAC

[0108] ATATATTCCTGTCTACAGATGGTCTCTTTTGGAATTGATTTTTGTTTCTAATTTGTTGGACA

[0109] GATGGTCTCTTTTGGAATTGATTTTTTTTCTAATTTGTTGGATATTTATTGGACTGGTGCAA

[0110] CAATTCATCCCCTCACACATGATTTGATCTATTCCATATACTCAGATGAATTGTTGGAATCC

[0111] TACTATTTCACCAGTATGGATCATGTTCAGATATTAGCTATTGACTTTTAGATATGACGTTTG

[0112] AAATATAAATTATTTTTTGTGACTTGTTTATTATTAAAGGTACATCAAACATAACTTATAATT

[0113] TTACATATTTGCACTATTTTTTTTTGTAAGGTGAATGGACAAACGTCATGTTCAAAAGTCA

[0114] GCAATGTCATATATTTAAAAAAGGAGGGAGGATTGCATTAGGAGTTCATAAGAGTGATAA

[0115] ACACGGCTTGTTAATAAGTGAATGATTATCTAACTTAACATATATATCATCCACAACAATGA

[0116] AAATACAGTCTATGCACACATTACTTTAACCAGTGTCTTGTACTAGCATCAATACTAACTA

[0117] GACTTTGCACTCTTTTGATGTCATTTCGTCTCAATGTTGTCATTTCAGAAATATCCATTTAA

[0118] CTGTTCATAAGTAACACTTTTCATTCTGTTAATTACTTTTTGAATCATATTCTGGTTGCATGC

[0119] TGCTCTACTGTCTGGAAATATTCCAATGTCAACAGAATGTGTATGTGCACACATGCAGCAT

[0120] AAATAGCTCCCAGAAAGTTTCAAATAAATTTTTATATTGGATGGAATTTGAACTGCATACTA

[0121] TAAATATTATCCTATGAATTGTAGGAGTTGTGTAATATATTTGTTATTTATATTTTTTTTAATCT

[0122] TCTGAAATCAAGGGTATGAATAATTTTTTTAGTGGAACTAGTGTAGTCTTTGTAAGTATTTG

[0123] GCCTTATTTCATTGTTAAAATCAATAGTTTTGTCTATATGAATTAGTGTAGTTTTTTACACTA

[0124] GTAGTTGCATATATACAAGGTGGTGTTTCAAAAGACGACTAATATGGCAAAATTTATGAGA

[0125] TTCACTATCCGTACAAATCCTGGATCTATATATTACAATCGCTTTGAGATGAATGCAAATTA

[0126] GGATGGGCACAATTTTTTCATAGCATGGTGGAATGGTTTCTCTTGTCACCATCGTTTTGAA

[0127] CTCATCTTTATTATGTGCCCGCTAATTACTCCCTCCTTTCTAAATTGATTATCATGTAATAGA

[0128] AATCAAAATTTCTTAAATTGATCATCATATAACTGCATGGAGACGGATTTCATCGGAATACA

[0129] ATTAATACAACATGGGAGAATGCGTGCATGCTAGGGTGGAGTAATTGGCATGGAGTTTTAA

[0130] TCGATGTATGCATTGTGGGAGAATGTGTGCATTGTGTCTTGATTGATGTGATTTAAATTATT

[0131] TTTTGGTCTTGGTGCGTAAATTTATATGATGATCAATTTGAGAAGGAGACAGTATATTTTAA

[0132] AACCGTTCTGTCTAAGTGTTCACACAAGATCACTGATTTTTTTTTTTGCCTTCGAATAAAC

[0133] AGAACACAAGCAGGCCTTGTAATATTTAGTATATATTCTTGTCATAGCTTTTCCCTACGAAT

[0134] CAAAATGAATATCCTGTCTCAAATTGAACGGTATGGCGGCTGTTTGACTAGTCTAGTTTT

[0135] CTGCTTGGAATTTGGAAATACCGTGGCCTGTTTGGTGGAGCTTTAGATTCTGATAATCAGC

[0136] TGTTTGGTAGCAAGCTTCTGAAAATCTGGAAATGCTCTGAAACCCAGCTTCTCCAGCTTC

[0137] TGGCTTCTTAGTTCAATTTTCAGAATCTGTAACTACAGATTCTCAGAAGCTGTGGACTGTT

[0138] TGGGACAGCTTCTAGCAGAAGTAGCTTTTGGGAAAAGTTGCAGCTGGAAGAAGCTCCTC

[0139] CAAACAGGGCCTGTCTAGCTGAGCAAGGACAATTATTGGGTGCACAGAGTGTACAACAT

[0140] GTTCAGGCCTTCAGGGTTGCACTGTTGCTTACTTCGATACACTCCTACCTAGTAAACACAA

[0141] AGAATTAATTGACTGACTGGTGGCCTGGTGGGGCTGACCGATTGAGTAATACACAGTACT

[0142] ACCATTATACTCTATGCATAATCTATTCGATCTCCCACACCTCCCTTCATATGAGGTTTACCT

[0143] GCTGTGACGCACGCAGTCATGTAATTTTTAATTATTTTGATAGAACATCATGTTGAACAATT

[0144] AACCTTTTTGAGCTGCTGTTTTTAACAGAGAGTAATTGTGAAGGCACGGGATCATCAGAG

[0145] GAAGAGCAAGACCCTAGTGACAAGCAGCTGAAGCACCAGCTTCTGAGGAAGTACGGTG

[0146] GCTCGTTGGGCGATCTCCGGCAGGTGTTCTCCAAGAGGACCAAGAAAGGGAAGCTTCCC

[0147] AAGGAGGCCAGGCAGAAGCTTCTGCACTGGTGGGAGCTGCACTACAAGTGGCCCTATCC

[0148] CTCCGTACGCACGCATCACATCCCGTTTGATCAATTCAACCACATTTTCTGCACCAAATTA

[0149] ACTCGTTTGAAAATGCGTGAAATTTACGGGGTTGTGTATGAGCAGGAGATGGAGAAGATG

[0150] ACGCTGGCGCAGACGACGGGGCTGGACCAGAAGCAGATCAACAACTGGTTCATCAACC

[0151] AGAGGAAGCGGCACTGGAAGCCGACGCCGGTGGCAGGCACGGCCTTCCCGACGATGGA

[0152] AGCTGCCGGAGGCGGCTTCCGACACTCCGGCCACGGCGGCGGCCTTGCGGCGGCGGCG

[0153] GCGCTGCCGCTGTACATGGGCAGGCCGTTCGTTGTGGACGGCATGTACCGGCTCGGGTCT

[0154] TGAAGCCTCTGGGGATCGCATGGTGTTTGCATGGTGTTTTTTGCTTGGTAAATATTTGCTA

[0155] GCAGCTTCCCATGATCAGCCTGTGGGATGGTGAAATGGAAGAACTGATGAATCTTCATCA

[0156] CGATCTTTCCTCTCATTTTTTTTTCTTTGGTGTGTGATAAAAGCTCAGATGTACCACGTACT

[0157] ACTGTCTGCTTCTTTGCAAATGGATATGTAATTGAAAGCTTTAATTCTAAACATGGGACTC

[0158] CTCCGTTTTTATATTATAAGTCGTTTTGATTTTTTTATTAAACTTGGTTAAGTTTAATTAATTT

[0159] TATAAAAAAATATAGCAATAATTTTAACACAAAACAAATACATCATCAAAAATTTTCAATG

[0160] TTACATATAATCAAATTAATTTGATGTTATAGTTGTTGCTGAATTTTCATATAAACTTAATCA

[0161] AACTTAAAGAAAATTGACTAAAAAGATTAAATTAATACATAATATAAAATGGAGGTAGTAT

[0162] ATTATTCTTGGGCGAGATAGCTCTAACGTCTA

[0163] SEQ ID NO.2OsKNOX8基因CDS序列:

[0164] ATGGAGAGCTTCGCCAGTCTCGCAGGAGGTGGCAGCAGCAGCACCACAGCTCGGTTGCC

[0165] GGAGCTGATCTCGCCGGAGAACCCGGACCACATCTCGCCGCCCCCGTTGCTGTACCAGCT

[0166] GCTCGCCGGCCCGGAGTCGTCGGCGCGGCAGCATGGGCATGATGGGCATCACCACGGCG

[0167] GCGGTGGCGGCGAAGCAGCAGCAGCAGCTGTGCAGGGACAGGTCTCGCCGGCAGGCGC

[0168] GGAGGCGGCGGTCAAGGCCGAGATCATGTCGCATCCACAGTACTCGGCTCTCCTAGCCG

[0169] CCTACTTAGGCTGCAAAAAGGTCGGGGCGCCGCCGGATGTACTGACCAAGCTCACGGCT

[0170] GTGCCGGCGGCGCAGCAGCTGGACGAAGCCGACGGCCACCCTCGTCGCCGGCACGAGC

[0171] CACAGCGTGATGACGACCCGGATCAGCTCGATCAGTTCATGGACGCGTACTGCAGCATGC

[0172] TGACGAGGTACAGGGAGGAGCTGGAGCGGCCGATCCTGGAAGCCGCGGAGTTCTTCAG

[0173] CAGGGTGGAGACGCAGCTCGATTCGCTCGCTGAGAGTAATTGTGAAGGCACGGGATCAT

[0174] CAGAGGAAGAGCAAGACCCTAGTGACAAGCAGCTGAAGCACCAGCTTCTGAGGAAGTA

[0175] CGGTGGCTCGTTGGGCGATCTCCGGCAGGTGTTCTCCAAGAGGACCAAGAAAGGGAAG

[0176] CTTCCCAAGGAGGCCAGGCAGAAGCTTCTGCACTGGTGGGAGCTGCACTACAAGTGGCC

[0177] CTATCCCTCCGAGATGGAGAAGATGACGCTGGCGCAGACGACGGGGCTGGACCAGAAGC

[0178] AGATCAACAACTGGTTCATCAACCAGAGGAAGCGGCACTGGAAGCCGACGCCGGTGGC

[0179] AGGCACGGCCTTCCCGACGATGGAAGCTGCCGGAGGCGGCTTCCGACACTCCGGCCACG

[0180] GCGGCGGCCTTGCGGCGGCGGCGGCGCTGCCGCTGTACATGGGCAGGCCGTTCGTTGTG

[0181] GACGGCATGTACCGGCTCGGGTCTTGA

[0182] SEQ ID NO.3基因OsKNOX8编码的蛋白质的氨基酸序列:

[0183] MESFASLAGGGSSSTTARLPELISPENPDHISPPPLLYQLLAGPESSARQHGHDGHHHGGGGG

[0184] EAAAAAVQGQVSPAGAEAAVKAEIMSHPQYSALLAAYLGCKKVGAPPDVLTKLTAVPAAQQ

[0185] LDEADGHPRRRHEPQRDDDPDQLDQFMDAYCSMLTRYREELERPILEAAEFFSRVETQLDSL

[0186] AESNCEGTGSSEEEQDPSDKQLKHQLLRKYGGSLGDLRQVFSKRTKKGKLPKEARQKLLH

[0187] WWELHYKWPYPSEMEKMTLAQTTGLDQKQINNWFINQRKRHWKPTPVAGTAFPTMEAAG

[0188] GGFRHSGHGGGLAAAAALPLYMGRPFVVDGMYRLGS

Claims

1. Use of a knockout vector for knocking out the OsKNOX8 gene, or reducing the expression of the protein encoded by the OsKNOX8 gene, or transferring the OsKNOX8 gene into rice for improving the salt tolerance of rice, characterized in that: The amino acid sequence of the protein encoded by the gene OsKNOX8 is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The gene OsKNOX8 is a DNA molecule as shown in 1) or 2) below: 1) A DNA molecule having a genomic sequence as shown in SEQ ID NO. 1; 2) A DNA molecule whose CDS sequence is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The knockout vector of the gene OsKNOX8 is a CRISPR-Cas9 vector targeting the target site shown in SEQ ID NO.

6.

4. The use according to claim 1, characterized in that The amplification primers for the gene OsKNOX8 are selected from Primer 1 shown in SEQ ID NO. 4 and Primer 2 shown in SEQ ID NO. 5, or Primer 5 shown in SEQ ID NO. 9 and Primer 6 shown in SEQ ID NO. 10.

Images